Protein fusions for the translocation of apo-protein into the periplasmic space

ABSTRACT

A method of protein synthesis which comprises providing a genetic unit comprising a nucleotide sequence coding for a pre-form apo-protein. The pre-form apo-protein is synthesized in a cytoplasmic region of a cell and translocated to the periplasmic region of the cell for conversion to a corresponding holo-protein.

FIELD OF THE INVENTION

The present invention is concerned with protein synthesis and inparticular with the production of recombinant fusions between cytochromeb₅ and foreign proteins which can be translocated out of the cell intothe periplasmic space of, for example, Escherichia coli.

BACKGROUND OF THE INVENTION

The mechanism by which a cytoplasmically-synthesised (recombinant)protein is translocated into the periplasmic space of Escherichia coliis known to occur by the workings of the signal hypothesis (see reviewby Stardler, J. A. and Silhavy, T. J., 1990, Methods in Enzymology,167-187). Many such proteins are initially synthesised as precursorforms carrying extensions at their amino termini known as signalsequences (peptides). The signal sequence has the information requiredfor selective translocation of the passenger part of the proteinmolecule across the cytoplasmic membranes of the bacteria. The signal iscleaved off soon after the periplasmically-deposited protein has gainedits native biological fold and function.

SUMMARY OF THE INVENTION

Using this approach we have developed a bacterial system which allowsproduction of chimeric or fusion forms of coloured proteins. Theseproteins are secreted into the periplasm where they are afforded greaterprotection against potential degradation.

According to a first aspect of the present invention there is provided amethod of protein synthesis which comprises provision of a genetic unitcomprising a nucleotide sequence coding for a pre-form of anapo-protein, synthesizing said pre-form apo-protein in a cytoplasmicregion of a cell, and translocating said synthesised pre-formapo-protein to the periplasmic region of said cell, so as to permitconstitution in said periplasmic region of a signal-processedapo-protein which can then be converted to the correspondingholo-protein.

According to a second aspect of the present invention, there is provideda genetic precursor unit which comprises a nucleotide sequence codingfor a pre-form apo-protein, said nucleotide sequence being such thatsaid pre-form apo-protein is translocatable from a cytoplasmic region ofa cell to the periplasmic region of said cell, so as to permitconstitution in said periplasmic region of a processed apo-protein andconversion into a corresponding holo-protein.

The genetic unit is preferably suitable for expression in thecytoplasmic region of a bacterial host cell, such as an E.coli hostcell. The preferred E.coli strain is TB-1, F ara Δ (lac-proAB) rps φ 80dlacZΔM15 hsdR17 (rk+_(m) ⁺ k)! or N4830-1 f-suo his-ilvgalK-(λch1D-pg1)(λBam N+CI857 H1)!.

A preferred apo-protein comprises a cytoplasmic cytochrome, whichtypically comprises the soluble core domain of cytochrome b₅ of liverendoplasmic reticulum.

Cytochrome b₅ of the endoplasmic reticulum of mammalian liver is a wellcharacterised, small haemoprotein of 16.7 kD which plays a central rolein a variety of electron transfer reactions related to fatty aciddesaturation, redox cycling of oestrogen and reduction of cytochromeP-450 reductase.

The hepatic cytochrome b₅ is composed of two domains, theabove-described soluble, enzymatically active, haem-containing globularcore (b₅) of about 12 kD and a smaller carboxy terminal tail anchored inthe microsomal membrane. Within the tail portion, a stretch of 23hydrophobic amino acid residues, also definable as an "insertion"sequence, autonomously and post-translationally integrates cytochrome b₅into the lipid bilayer of the endoplasmic reticulum such that the activecore domain is laterally disposed from the reticulum facing thecytoplasm.

It is preferred that the genetic unit further comprises a nucleotidesequence which codes for an amino-terminal signal peptide (the preregion of pre-form apo-protein) which is recognised by the cell todirect the pre-form apo-protein to the cytoplasmic membrane and thencesubsequently translocated into the periplasm of the cell. The signalpeptide typically comprises E.coli alkaline phosphatase, which isadvantageously in linkage with cytochrome b₅ (apo-protein). The methodtherefore preferably further comprises the step of directing theapo-protein to the cytoplasmic membrane, under the direction of thesignal peptide, so as to facilitate the above-described translocation ofthe pre-form apo-protein from the cytoplasm to the periplasmic space ofthe bacterium.

In this preferred embodiment of the present invention where the geneticunit further comprises a nucleotide sequence which codes for anamino-terminal signal peptide, the invention can be used to investigatesignal peptidase which in vivo proteolytically cleaves N-terminal signalpeptides.

Many proteins which are synthesised in the cytosol have to be targetedto various extracellular and subcellular locations. This involvestranslocation across one or more lipid bilayers impermeable to largehydrophilic molecules. A feature of most translocated proteins is thatthey are synthesised as precursor forms carrying N-terminal signalsequences, which as discussed above are proteolytically cleaved bysignal peptidase, either during or immediately after transport of thepassenger protein across the biological membrane.

Signal peptidase is highly specific as it cleaves only preproteinprecursors. Although lacking sequence homologies, signal sequences arefunctionally highly conserved. Despite significant variations in theirlengths, all signals have three definable regions, namely an N-terminalregion carrying one or two basic residues, a middle core encompassinghydrophobic residues and a terminal region specifying the cleavage site.

The conventional assay for signal peptidase has until now relied on theuse of natural precursor proteins which are synthesised in a cell-freetranslation system in a highly radioactive form. The extent ofprocessing is monitored by incubating the radio-labelled substrate witheither membranes or signal peptidase and monitoring the processed bandsby autoradiography following gel electrophoresis. The assay is not onlytime-consuming and expensive but suffers from the use of minusculequantities (fmol) of substrates in relation to enzyme quantities, withinevitable contamination by many cellular components derived from theuse of in vitro synthesis systems.

We have now overcome the above problems, and there is further providedby the present invention an assay system for signal peptidase, whichassay system comprises:

(a) a genetic precursor unit which comprises a nucleotide sequencecoding for an apo-protein and an amino-terminal signal peptide, thenucleotide sequence being such that the apo-protein is translocatablefrom a cytoplasmic region of a cell to the periplasmic region of thecell, so as to permit constitution in said periplasmic region of aprocessed apo-protein; and

(b) signal peptidase, a source thereof or a precursor therefor, whereinthe signal peptidase can effect proteolytic cleavage of the aminoterminal signal peptide from the apo-protein.

The genetic unit of the assay system is substantially as hereinbeforedescribed and is advantageously suitable for expression in a bacterialmedium such as E.coli and the assay system preferably further comprisesa host medium such as E.coli substantially as hereinbefore described.

Preferably the assay system further comprises means for monitoring theproteolytic cleavage of the amino terminal signal peptide from theapo-protein. The monitoring means typically comprises means forseparating the cleaved apo-protein from the pre-form apo-protein, andmeans for spectrophotometrically monitoring the cleaved apo-proteinfollowing supplementation thereof with exogenous haem. Typically thepre-form apo-protein is phase-separated from the cleaved apo-protein,the pre-form only being soluble as an aggregate in the presence of adetergent, the cleaved apo-protein being water soluble.

The provision of such an assay system is advantageous in investigatingthe mechanistic and structural properties of signal peptidase. Aparticularly useful application of an assay system according to thepresent invention is in the identification of inhibitors of signalpeptidase which can subsequently be used as probes in investigating thereaction mechanism of signal peptidase.

There is further provided by the present invention a method ofidentifying inhibitors to signal peptidase, which method comprisesproviding an assay system substantially as described above, introducinga test material into cells of a host medium used in the assay system,and monitoring the effect of said test material on a selected activityof the signal peptidase.

Advantageously, the identification method involves monitoring thecleavage of the amino-terminal signal peptide from the apo-protein byspectrophotometric means substantially as hereinbefore described.

Preferably the test material comprises any of the following: a consensussignal peptide (which typically comprises a 19 residue peptide), one ormore transition metal ions, and carboxy-modifying reagents.

Signal peptidase is a known crucial rate limiting step in the pathway ofprotein secretion and can offer a locus for control or regulation of anessential biofunction, namely protein export. Thus the identification ofa signal peptidase inhibitor can provide a tool to understand thereaction mechanism of signal peptidase, and also, more importantly, ameans to regulate growth of microorganisms and protein secretorydisorders in higher organisms.

The invention allows the observation of the effects of competitiveinhibition by selected inhibitors in order to identify the preciseregion of signal sequence that is essential for recognition/cleavage bysignal peptidase.

There is further provided by the present invention a method ofrecognising an active binding site of signal peptidase, which methodcomprises contacting the signal peptidase with at least onphotoactivatable form of a radio-labelled (typically radioiodinated)inhibitor of the peptidase, and identifying the binding location of theinhibitor to the peptidase.

There is further provided by the present invention, a test materialidentified as an inhibitor to signal peptidase, for use in modulating invivo protein secretion.

The test material may advantageously comprise a synthetic inhibitor ofsignal peptidase, and has beneficial applications as a biocidal agent,regulating secretory disorders in mammalian cells, control of plantgrowth and the like.

The genetic unit typically comprises a plasmid having a vector as shownin any of FIGS. 1 to 3, or a DNA sequence as shown in any of FIGS. 4 to6 (SEQ ID NOS 1 to 3, respectively). The most significant features ofthe plasmid sequences are as follows:

a) a native pho promoter for transcriptional control. The pho promoteris available coded for by the plasmids pSEC-cyt which is inducible bygrowth of the bacteria in a phosphate-limited medium;

b) a signal sequence coding for alkaline phosphatase as hereinbeforedescribed; and

c) a soluble cytochrome b₅ core gene sequence.

There is further provided by the present invention a genetic unit havinga vector substantially as illustrated in any of FIGS. 1 to 3, or a DNAsequence as illustrated in any of FIGS. 4 to 6 (SEQ ID NOS 1 to 3respectively), which is capable of generating translocated formsnon-fusion (FIG. 1 or 4) or fusion (chimeric) forms of colouredcytochrome b₅ (FIGS. 2,5 and 3,6). The foreign protein can be tagged tothe cytochrome b₅ protein at its carboxy (C) FIGS. 2,5) or amino (N)terminus (FIGS. 3,6).

The pho promoter is typically capable of minimising the lethality of theexpressed chimera to the host by maintaining the expression of chimeraunder the tight transcriptional control of the native pho promoter, inplasmids pSEC-cyt/C and pSEC-cyt/N, inducible only by growth of thebacteria in a phosphate limited medium. In pSEC-cyt/C fusions areintroduced by linkage of a foreign DNA sequence at the carboxy terminusof cytochrome b₅ gene through the unique BclI site. In pSEC-cyt/Nfusions are introduced immediately at the N-terminus of cytochrome b₅but after the signal sequence, through the unique BamHI and/or PstIsites.

The method of expression of the pre-form cytochrome b₅ or its chimericderivative typically involves initiation of transcription, and henceprotein synthesis, by control of the incubation conditions of the hostmedium. Typically the initiation stage comprises cultivation of thebacterial host in a phosphate-limited medium (0.1 mM phosphate), andspectrophotometric or visual monitoring (transformation of the colour ofthe bacterial cells from a grey to pink colour) of the synthesiscytochrome b₅ (processed) or its chimera (processed) in E.coliharbouring pAA-cyt or pSEC-cyt, respectively, under the transcriptionalcontrol of the pho promoter.

It is further preferred that the method involves translocation of aprosthetic group from the cytoplasmic region of the cell to theperiplasmic region, for combination with the signal-processed apo-formof the chimeric protein in the periplasmic region to form theholo-protein as hereinbefore described. It is preferred that theapo-protein is first translocated to the periplasmic region, followed bysubsequent translocation of the prosthetic group, such that theapo-protein can act as a sink for the latter. In the case where theapo-protein comprises the cytochrome b₅ soluble core, the prostheticmaterial comprises the haem (protoporphyrin IX) prosthetic group. Theperiplasmic accrual of the apo cytochrome b₅ core is thought to triggerthe subsequent translocation of the haem-prosthetic group.

It is envisaged that a prosthetic material may be present in theperiplasmic region of the cell, although this does not appear to be thecase with cytochrome b₅ due to the observed build up of periplasmicapo-cytochrome b₅ and ensuing time lag preceding the appearance ofperiplasmic holo-cytochrome b₅.

Production of the exported periplasmically-localised haemoprotein cantypically exceed 6 mg/liter of culture after five hours induction.Mutated forms of the cytochrome b₅ have been developed which can enhanceexportation of the mature forms by more than tenfold. The approach maybe useful for the production and secretion of coloured forms and fusionsin eukaryotic cells.

The method according to the invention typically further comprisesmonitoring of protein synthesis by observing the visual transformationof the bacterial cells from a grey translucent to a bright pink colour.This colour change is due to the expression of the cytochrome b₅ gene.The monitoring may be carried out by spectrophotometric means where anaccurate analysis of the extent of protein synthesis is required, soallowing the protein synthesis to be terminated when sufficient proteinhas been synthesised.

The above-described method allows a prokaryotic signal sequence to beappended with cytochrome b₅, such that this natural cytoplasmic proteinof eukaryotic origin is transformed into a secretory form in E.coli.Furthermore, the expression of the eukaryotic gene encoding the pre-formapo-protein or its chimera, synthesised in the cytoplasm, is such thatthe post-translocationally processed component in the periplasm givesrise to the holo-protein or its fusion counterpart.

According to the present invention there is further provided aproteinaceous material comprising a pre-form apo-protein which issynthesised in the cytoplasmic region of a cell, the pre-formapo-protein being translocatable to the periplasmic region of said cell,so as to allow constitution in said periplasmic region of acorrespondingly processed apo-protein, the binding of a prosthetic group(typically the haem group) to the apo-protein forms the holo-protein.

There is further provided by the present invention an apo-proteinsubstantially as hereinbefore described, in combination with a signalpeptide for selective translocation (export) or secretion of saidapo-protein from the cytoplasmic to the periplasmic region of a cell, soas to allow constitution in the periplasmic region of a correspondingholo-protein from said apo-protein.

According to yet a further aspect of the present invention there isprovided a holo-protein constituted in the periplasmic region of a cellfrom an apo-protein, or by a method, substantially as hereinbeforedescribed. There is further provided a holo-protein derived from agenetic precursor unit, substantially as illustrated with reference toany of FIGS. 4 to 6.

The above-described apo- and holo-proteins typically respectivelycomprise the apo- and holo- forms of cytochrome b₅ or its respective N-or C-terminally fused chimeric forms. The fusions can be generated ateither the amino or carboxy terminus of the secreted recombinantprotein.

There is further provided by the present invention an E.coli host mediumcontaining a genetic precursor unit substantially as hereinbeforedescribed.

There is further provided a kit for use in protein synthesis, which kitcomprises:

a) a bacterial host medium substantially as hereinbefore described; and

b) genetic material comprising a nucleotide sequence coding for apre-form apo-protein, such that said pre-form apo-protein coded for bysaid nucleotide sequence is translocatable from the cytoplasmic to theperiplasmic region of a cell of the host medium, so as to allowconstitution in the periplasmic region of a holo-protein from theapo-protein.

The kit typically comprises a bacterial host medium, such as E.coli,together with genetic material comprising precursor constituent genessuitable to be included in a genetic unit substantially as hereinbeforedescribed.

An example of a typical synthesis of a fusion protein according to thepresent invention will now be described.

A gene coding for a foreign protein or peptide was fused in a readingframe from either (i) the carboxy-terminus or (ii) the N-terminus of thesoluble core of cytochrome b₅. The foreign gene was ligated betweeneither (i) the unique BclI restriction site and the downstreampolylinker site or (ii) the unique PstI and BamHI site located betweenthe signal sequence and cytochrome b₅.

E.coli TB-1 (Lac) cells were then transformed with the ligation mixture.

Transformants were plated out on Luria broth agar (1.5% (w.v)) platescontaining ampicillin (75 μg/ml), isopropyl-β-D-galactopyranoside (IPTG)(100 μM),5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) (20 μg/ml).Transformant colonies bearing potential inserts were white in colourwhereas non-recombinants were blue in colour. If required, therecombinant plasmids could be further verified by restriction mapping.

For induction, a starter bacterial culture cultivated to saturation inLB/ampicillin was applied at a 2% (v/v) inoculum into MOPS medium. Thecultures were grown with shaking at 100 revs/min at 35° C. for 5 to 10h.

The following table indicates the composition of the MOPS medium. Allsolutions were sterilised by autoclaving or filter sterilisation. Thefinal concentration of phosphate in the MOPS medium was about 0.1 mM.

    ______________________________________    Component       % (v/v) in MOPS    ______________________________________    Water           74.5    M.sup.a         20    O.sup.f         0.2    P.sup.a         0.01    S.sup.a         0.1    VFCAA.sup.a     2    20% glucose.sup.f                    1    0.05% thiamine B1.sup.f                    0.2    25 mg/ml ampicillin.sup.f                    0.3    ______________________________________     M, 4.2% (w/v) MOPS buffer, 0.4% (w/v) Tricine, 1.46% NaCl, 0.8% KOH,     0.2555% NH.sub.4 Cl     O, 5.36% MgCl.sub.2, 0.16% HCl, 0.1% FeCl.sub.2, 0.00368%     CaCl.sub.2.2H.sub.2 O, 0.00128% H.sub.3 BO.sub.3, 0.0008%     MnCl.sub.2.4H.sub.2 O, 0.00036% CoCl.sub.2.H.sub.2 O, 0.00008%     CuCl.sub.2.2H.sub.2 O, 0.0068% ZnCl.sub.2, 0.0121% Na.sub.2     MoO.sub.4.2H.sub.2 O.     P, 1.0M KH.sub.2 PO.sub.4     S, 0.276M K.sub.2 SO.sub.4     VFCAA, 7.5% vitaminfree Casamino acids     .sup.a autoclaved prior to use     .sup.f filter sterilised prior to use

Positive expression of the fusion protein was indicated by pink colourof the bacterial cells obtained by centrifugation at 5,000×g for 5 min.

The protein was extracted from the bacteria by the following "osmoticshock" method:

(i) The cells (derived from a 30 ml culture) were gently suspended in 1ml of 20% sucrose, 0.3 M Tris-HCl (pH 8), 1 mM Na₂ EDTA)(STE) andincubated at room temperature (22° C.) for 10 min.

(ii) The cells were then harvested as pellet by centrifugation at5,000×g for 6 min and thoroughly resuspended in residual STE volume.

(iii) The plasmolysed cells were osmotically shocked by rapidlysuspending in ice-cold 0.5 mM MgCl₂ and left on ice for 10 min.

Centrifugation at 10,000×g for 10 min yielded supernatant comprising theperiplasmic fraction. Pink coloration of the supernatant implied thatfusion protein had been efficiently extracted from the periplasmicspace.

The quantity of the recombinant protein was monitored by subjecting afraction of the extract to a spectral scan from 350 to 500 nm. Theoxidised cytochrome was identifiable by the rise in absorbance at 413nm. Often a significant pool of the apo-protein may be present. This maybe converted to holo cytochrome by addition of haem to the supernatantto a final concentration ranging from 5 to 20 μM; an equivalent amountof haem was added to the blank cuvette to counteract the absorbance riseby excess haem in the sample. The amount of protein may be calculatedfrom the mM extinction coefficient of the oxidised cytochrome b₅ of 115and 413 nm.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will now be further illustrated, by way of exampleonly, by reference to the accompanying FIGS. 1 to 6, wherein:

FIGS. 1,2 and 3 illustrate expression vectors pAA-cyt, pSEC-cyt/C andPSEC-cyt/N of a genetic unit according to the present invention;

FIGS. 4-6 illustrate the genetic sequences of the vectors respectivelyillustrated in FIGS. 1-3 (SEQ ID NOS 1 to 3, respectively); and

FIGS. 7-9 schematically illustrate modifications of plasmids accordingto the present invention, which involve cloning, expression andisolation of the chimeric proteinaceous material.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 illustrates plasmid PAA-cyt constructed for the periplasmicproduction of non-fusion, secreted holo-cytochrome b₅.

FIG. 2 illustrates the plasmid PSEC-cyt/C constructed for linkage at the3' terminus (C-terminus) of the soluble cytochrome b₅ core gene with aforeign gene sequence, according to the present invention.

FIG. 3 illustrates the plasmid PSEC-cyt/N for linkage at 5' terminus,between the C-terminus of alkaline phosphatase signal and the N-terminusof cytochrome b₅ of the cytochrome b₅ core gene, according to thepresent invention.

Referring now to FIGS. 1 to 3, the essential features of the illustratedvector are as follows:

Pho, Pho promoter activated in bacteria grown on phosphate limitedmedium;

S/D), Shine Dalgarno sequence for translation initiation of alkalinephosphatase signal;

SS, alkaline phosphatase signal sequence gene encoding 21 residue signalincluding the first arginine residue of mature alkaline phosphatase;

Cyt, soluble core of cytochrome b₅ ;

T, tail of the native cytochrome b₅ ;

Lac, Lactose operon promoter; and

PLS, polylinker sites: EcoRI, (Hind III), SacI, KpnI, SmaI, BamHI, XbaI,SalI, HincII, BspMI, PstI, SphI, HindIII. (In the polylinker sites,EcoRI and HindIII can be in the reverse order to that shown.)

SS-cyt/C, codes for the complete precursor of the cytochrome b₅including the tail portion of the haemoprotein.

Referring now to FIG. 7, the figure illustrates the scheme of appendageof alkaline phosphatase signal to rat liver ER cytochrome b₅ in plasmidconstructions pAA-cyt, pSEC-cyt/C and pSEC-cyt/N. The full-lengthsynthetic cytochrome b₅ gene, contained within the PstI-EcoRI sites andcarrying an in-built ribosomal binding site, was cloned within thepolylinker site of pBluescript SK+ to give pBLUE-cyt. In step 1, theunique PstI site, located upstream of the Shine/Dalgarno sequence ofcytochrome b₅ gene was displaced immediately adjacent to the cytochromeb₅ methionine initiator codon by replacing PstI-AatII deletion with the27 bp synthetic oligonucleotide duplex. This gave plasmid pA-cyt. Icyt.In step 2, insertion of the modified BamHI-HindIII-cleaved cytochrome b₅gene (from pA-cyt) into identically-cleaved pFOG402. This yieldedplasmid pAF-cyt in which cytochrome b₅ is placed under the control ofphoA promoter but in an unphased reading frame with the alkalinephosphatase signal sequence. In step 3, involving construction ofpAA-cyt, it shows the generation of the correct reading frame betweenthe signal sequence and cytochrome b₅ by flush-ending PstI-BamHI-cleaved PAF-cyt and recircularising the plasmid. In this process the 21residue signal sequence was installed in a contiguous reading frame withthe mammalian cytochrome b₅ such that the +1 arginyl residue of maturealkaline phosphatase became included. This was to enhance the exportproperties of the cytochrome b₅ apo-protein. To minimise lethality tothe host, the chimera was placed under tight transcriptional control ofthe native pho promoter in plasmid pAA-cyt, inducible by growth of thebacteria in a phosphate-limited medium. In step 4, the plasmid wasfurther modified by appending the tail portion (T) of cytochrome b₅.Finally, translocation of EcoO109-cleaved cytochrome b₅ -T segment intoidentically cleaved PUC19 gave plasmid PSEC-cyt/C.

Plasmid PSEC-cyt/N was derived by excising EcoRI-HindIII fragment fromPAF-cyt containing pho-A-ss-b₅ and cloning into identically cleavedpUC19.

Referring now to FIG. 8, an expression vector as illustrated in FIG. 2was modified by appending in fusion at the C-terminus of cytochrome b₅with a gene sequence coding for the transit peptide of nitrite reductase(step 1). The modified plasmid (pCYT-nTP) was introduced into thecytoplasmic region of E.coli TB-1 (Lac) cells and the correspondingchimeric protein comprising the cytochrome b₅ apo-protein fused at itsC-terminus with nitrite reductase transit peptide (pre-form apo-form ofcytochrome b₅ -nitrite reductase transit peptide), was expressed in thecytoplasm (step 2). The pre-form chimeric protein was processed (apoform of cytochrome b₅ -nitrite reductase transit peptide) and afterbeing translocated into the periplasmic region of the E.coli cells (step3), it combined with the haem-prosthetic group to give the holo-protein(holo form of cytochrome b₅ -nitrite reductase transit peptide).

The crude recombinant chimeric protein was extracted from the pinkbacteria (20 ml culture) by an osmotic shock technique (step 4). Thetechnique involved suspending the cells in 1 ml of 20% sucrose, 0.3MTris-HCl (pH 8), 1 mM Na₂ EDTA (STE) and incubation at room temperaturefor ten minutes. The cells were harvested as pellet by centrifugation at5,000 g for six minutes and resuspended in residual STE volume. Theplasmolysed cells were osmotically shocked by suspending in a minimumpossible volume (approximately 5 ml) of ice-cold 0.5 mM MgCl₂ and lefton ice for 10 minutes, followed by centrifugation at 10,000 g for tenminutes to yield a supernatant comprising the periplasmic fraction ofthe cells.

The chimera traced by its bright pink colour was purified by asingle-step affinity chromatography (step 5), such as DEAE Sepharosecolumn chromatography or haem-affinity chromatography. The chimera wasthen selectively cleaved (step 6) by CNBr treatment to generateauthentic nitrite reductase transit peptide.

The 21 residue signal sequence is installed in a contiguous readingframe with the mammalian cytochrome b₅, the +1 arginyl residue of maturealkaline phosphatase being included to enhance the export properties ofthe apo-protein chimera.

Referring now to FIG. 9, an expression vector as illustrated in FIG. 3was modified by inserting in a contiguous reading between the signalsequence and cytochrome b₅, a BamHI-PstI synthetic gene coding fornitrite reductase transit peptide (step 1). The derivative plasmid(pnTP-cyt) was introduced into the cytoplasmic region of E.coli TB-1(Lac) cells and the corresponding chimeric protein comprising thepre-form apo-form of nitrite reductase transit peptide-cytochrome b₅ wasexpressed in the cytoplasm (step 2). The processed form of the chimericprotein (apo-form of nitrite reductase transit peptide-cytochrome b₅)was then translocated into the periplasmic region of the E.coli cells(step 3), where combination with the haem-prosthetic group occurredconstituting the holo-protein.

The crude recombinant protein was extracted from the pink bacterial (20ml culture) by an osmotic shock technique (step 4). The techniqueinvolved suspending the cells in 1 ml of 20% sucrose, 0.3M Tris-HCl (pH8), 1 mM Na₂ EDTA (STE) and incubation at room temperature for tenminutes. The cells were harvested as pellet by centrifugation at 5,000 gfor 6 minutes and resuspended in residual STE volume. The plasmolysedcells were osmotically shocked by suspending in a minimum possiblevolume (approximately 5 ml) of ice-cold 0.5 mM MgCl₂ and left on ice for10 minutes, followed by centrifugation at 10,000 g for ten minutes toyield a supernatant comprising the periplasmic fraction of the cells.

The chimera traced by its bright pink colour was purified by asingle-step affinity chromatography (step 5), such as DEAE Sepharosecolumn chromatography or haem-affinity chromatography. The nitritereductase transit peptide was cleaved from the isolated chimera by CNBr.

The 21 residue signal sequence is installed in a contiguous readingframe with the foreign gene and the subsequent mammalian cytochrome b₅.The +1 arginyl residue of mature alkaline phosphatase being included toenhance the export and processing characteristics of the chimericapo-protein.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 3    - (2) INFORMATION FOR SEQ ID NO: 1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 495 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: circular    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (vi) ORIGINAL SOURCE:    #coli     (A) ORGANISM: Escherichia              (B) STRAIN: TB-1    #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - GTCAGTAAAA AGTTAATCTT TTCAACAGCT GTCATAAAGT TGTCACGGCC GA - #GACTTATA      60    - GTCGCTTTGT TTTTATTTTT TAATGTATTT GTACATGGAG AAAATAAAGT GA - #AACAAAGC     120    - ACTATTGCAC TGGCACTCTT ACCGTTACTG TTTACCCCTG TGACAAAAGC CC - #GGATGGCT     180    - GAACAAAGCG ACAAAGACGT CAAATACTAC ACTCTGGAAG AAATCCAAAA AC - #ACAAAGAC     240    - TCGAAGTCGA CGTGGGTGAT CCTGCACCAT AAAGTATACG ACCTAACTAA AT - #TCCTCGAA     300    - GAGCACCCCG GGGGCGAAGA AGTCCTGAGA GAACAGGCCG GCGGTGACGC GA - #CTGAAAAC     360    - TTCGAAGACG TTGGCCATAG TACCGACGCT CGAGAACTGT CGAAAACGTA CA - #TCATCGGT     420    - GAGCTGCACC CGGACGATCG TTCTAAAATC GCGAAACCGT CCGAAACTCT GT - #AATGAGAA     480    #   495    - (2) INFORMATION FOR SEQ ID NO: 2:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 3918 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: circular    -     (ii) MOLECULE TYPE: DNA (genomic)    #2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GA - #GACGGTCA      60    - CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG TC - #AGCGGGTG     120    - TTGGCGGGTG TCGGGGCTGG CTTAACTATG CGGCATCAGA GCAGATTGTA CT - #GAGAGTGC     180    - ACCATATGCG GTGTGAAATA CCGCACAGAT GCGTAAGGAG AAAATACCGC AT - #CAGGCGCC     240    - ATTCGCCATT CAGGCTGCGC AACTGTTGGG AAGGGCGATC GGTGCGGGCC TC - #TTCGCTAT     300    - TACGCCAGCT GGCGAAAGGG GGATGTGCTG CAAGGCGATT AAGTTGGGTA AC - #GCCAGGGT     360    - TTTCCCAGTC ACGACGTTGT AAAACGACGG CCAGTGAATT CGAGCTCGGT AC - #CCGGGGAT     420    - CCTCTAGAGT CGACCTGCAG GCATGCAAGC TTGGCGTAAT CATGGTCATA GC - #TGTTTCCT     480    - GTGTGAAATT GTTATCCGCT CACAATTCCA CACAACATAC GAGCCGGAAG CA - #TAAAGTGT     540    - AAAGCCTGGG GTGCCTAATG AGTGAGCTAA CTCACATTAA TTGCGTTGCG CT - #CACTGCCC     600    - GCTTTCCAGT CGGGAAACCT GTCGTGCCAG CTGCATTAAT GAATCGGCCA AC - #GCGCGGGG     660    - AGAGGCGGTT TGCGTATTGG GCGCTCTTCC GCTTCCTCGC TCACTGACTC GC - #TGCGCTCG     720    - GTCGTTCGGC TGCGGCGAGC GGTATCAGCT CACTCAAAGG CGGTAATACG GT - #TATCCACA     780    - GAATCAGGGG ATAACGCAGG AAAGAACATG TGAGCAAAAG GCCAGCAAAA GG - #CCAGGAAC     840    - CGTAAAAAGG CCGCGTTGCT GGCGTTTTTC CATAGGCTCC GCCCCCCTGA CG - #AGCATCAC     900    - AAAAATCGAC GCTCAAGTCA GAGGTGGCGA AACCCGACAG GACTATAAAG AT - #ACCAGGCG     960    - TTTCCCCCTG GAAGCTCCCT CGTGCGCTCT CCTGTTCCGA CCCTGCCGCT TA - #CCGGATAC    1020    - CTGTCCGCCT TTCTCCCTTC GGGAAGCGTG GCGCTTTCTC AATGCTCACG CT - #GTAGGTAT    1080    - CTCAGTTCGG TGTAGGTCGT TCGCTCCAAG CTGGGCTGTG TGCACGAACC CC - #CCGTTCAG    1140    - CCCGACCGCT GCGCCTTATC CGGTAACTAT CGTCTTGAGT CCAACCCGGT AA - #GACACGAC    1200    - TTATCGCCAC TGGCAGCAGC CACTGGTAAC AGGATTAGCA GAGCGAGGTA TG - #TAGGCGGT    1260    - GCTACAGAGT TCTTGAAGTG GTGGCCTAAC TACGGCTACA CTAGAAGGAC AG - #TATTTGGT    1320    - ATCTGCGCTC TGCTGAAGCC AGTTACCTTC GGAAAAAGAG TTGGTAGCTC TT - #GATCCGGC    1380    - AAACAAACCA CCGCTGGTAG CGGTGGTTTT TTTGTTTGCA AGCAGCAGAT TA - #CGCGCAGA    1440    - AAAAAAGGAT CTCAAGAAGA TCCTTTGATC TTTTCTACGG GGTCTGACGC TC - #AGTGGAAC    1500    - GAAAACTCAC GTTAAGGGAT TTTGGTCATG AGATTATCAA AAAGGATCTT CA - #CCTAGATC    1560    - CTTTTAAATT AAAAATGAAG TTTTAAATCA ATCTAAAGTA TATATGAGTA AA - #CTTGGTCT    1620    - GACAGTTACC AATGCTTAAT CAGTGAGGCA CCTATCTCAG CGATCTGTCT AT - #TTCGTTCA    1680    - TCCATAGTTG CCTGACTCCC CGTCGTGTAG ATAACTACGA TACGGGAGGG CT - #TACCATCT    1740    - GGCCCCAGTG CTGCAATGAT ACCGCGAGAC CCACGCTCAC CGGCTCCAGA TT - #TATCAGCA    1800    - ATAAACCAGC CAGCCGGAAG GGCCGAGCGC AGAAGTGGTC CTGCAACTTT AT - #CCGCCTCC    1860    - ATCCAGTCTA TTAATTGTTG CCGGGAAGCT AGAGTAAGTA GTTCGCCAGT TA - #ATAGTTTG    1920    - CGCAACGTTG TTGCCATTGC TACAGGCATC GTGGTGTCAC GCTCGTCGTT TG - #GTATGGCT    1980    - TCATTCAGCT CCGGTTCCCA ACGATCAAGG CGAGTTACAT GATCCCCCAT GT - #TGTGCAAA    2040    - AAAGCGGTTA GCTCCTTCGG TCCTCCGATC GTTGTCAGAA GTAAGTTGGC CG - #CAGTGTTA    2100    - TCACTCATGG TTATGGCAGC ACTGCATAAT TCTCTTACTG TCATGCCATC CG - #TAAGATGC    2160    - TTTTCTGTGA CTGGTGAGTA CTCAACCAAG TCATTCTGAG AATAGTGTAT GC - #GGCGACCG    2220    - AGTTGCTCTT GCCCGGCGTC AATACGGGAT AATACCGCGC CACATAGCAG AA - #CTTTAAAA    2280    - GTGCTCATCA TTGGAAAACG TTCTTCGGGG CGAAAACTCT CAAGGATCTT AC - #CGCTGTTG    2340    - AGATCCAGTT CGATGTAACC CACTCGTGCA CCCAACTGAT CTTCAGCATC TT - #TTACTTTC    2400    - ACCAGCGTTT CTGGGTGAGC AAAAACAGGA AGGCAAAATG CCGCAAAAAA GG - #GAATAAGG    2460    - GCGACACGGA AATGTTGAAT ACTCATACTC TTCCTTTTTC AATATTATTG AA - #GCATTTAT    2520    - CAGGGTTATT GTCTCATGAG CGGATACATA TTTGAATGTA TTTAGAAAAA TA - #AACAAATA    2580    - GGGGTTCCGC GCACATTTCC CCGAAAAGTG CCACCTGACG TCTAAGAAAC CA - #TTATTATC    2640    - ATGACATTAA CCTATAAAAA TAGGCGTATC ACGAGGCCCT TTCGTCTTCA AG - #AATTCTCA    2700    - TGTTTGACAG CTTATCATCG ATAAGCTAGC TTTGGAGATT ATCGTCACTG CA - #ATGCTTCG    2760    - CAATATGGCG CAAAATGACC AACAGCGGTT GATTGATCAG GTAGAGGGGG CG - #CTGTACGA    2820    - GGTAAAGCCC GATGCCAGCA TTCCTGACGA CGATACGGAG CTGCTGCGCG AT - #TACGTAAA    2880    - GAAGTTATTG AAGCATCCTC GTCAGTAAAA AGTTAATCTT TTCAACAGCT GT - #CATAAAGT    2940    - TGTCACGGCC GAGACTTATA GTCGCTTTGT TTTTATTTTT TAATGTATTT GT - #ACATGGAG    3000    - AAAATAAAGT GAAACAAAGC ACTATTGCAC TGGCACTCTT ACCGTTACTG TT - #TACCTGTA    3060    - AAATCCTGTG ACAAAAGCCC GGATGGCTGA ACAAAGCGAC AAAGACGTCA AA - #ATAAAGTA    3120    - AGGAAATACT ACACTCTGGA AGAAATCCAA AAACACAAAG ACTCGAAGTC GA - #CGTTTGGG    3180    - ATTGGGTGAT CCTGCACCAT AAAGTATACG ACCTAACTAA ATTCCTCGAA TA - #ATATGGAG    3240    - CACCCCGGGG GCGAAGAAGT CCTGAGAGAA CAGGCCGGCG GTGACGGGGG AA - #GGGAAGGA    3300    - GCGACTGAAA ACTTCGAAGA CGTTGGCCAT AGTACCGACG CTCGAGAACT GT - #CGAAAACG    3360    - TACATCATCG GTGAGCTGCA CCCGGACGAT CGTTCTAAAA TCGCGAAACC GT - #CCGAAACT    3420    - CTGATCACTA CCGTTGAATC GAACTCTAGT TGGTGGACTA ACTGGGTTAT CC - #CTGCGATC    3480    - TCTGCTCTGG TTGTAGCGCT GATGTACCGT CTGTACATGG CTGAAGATTA AT - #GAAAATTA    3540    - GTTAAGAGAA TTCGATATCA AGCTTTAGTT CGTCAAGGCT TGGCTAAAGT TG - #CTTATGTT    3600    - TACAAACCTA ACAATACATA TGAACAACAT TTAAGAAAAA GTGAAGCACA AG - #CGAAAAAA    3660    - GAGAAATTAA ATATTTGGAG CGAAGACAAC GCTGATTCAG GTCAATAATG CT - #CATTGTAA    3720    - AAGTGTCACT GCTGCTAGTG GCACTTTTAT AATTTTTAGA TCCTCTACGC CG - #GACGCATC    3780    - GTGGCCGGCA TCACCGGCGC CACAGGTGCG GTTGCTGGCG CCTATATCGC CG - #ACATCACC    3840    - GATGGGGAAG ATCGGGCTCG CCACTTCGGG CTCATGAGCG CTTGTTTCGG CG - #TGGGTATG    3900    #3918              TC    - (2) INFORMATION FOR SEQ ID NO: 3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 511 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: circular    -     (ii) MOLECULE TYPE: DNA (genomic)    #3:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - GTCAGTAAAA AGTTAATCTT TTCAACAGCT GTCATAAAGT TGTCACGGCC GA - #GACTTATA      60    - GTCGCTTTGT TTTTATTTTT TAATGTATTT GTACATGGAG AAAATAAAGT GA - #AACAAAGC     120    - ACTATTGCAC TGGCACTCTT ACCGTTACTG TTTACCCCTG TGACAAAAGC CC - #GGATCCCC     180    - CGGGCTGCAG ATGGCTGAAC AAAGCGACAA AGACGTCAAA TACTACACTC TG - #GAAGAAAT     240    - CCAAAAACAC AAAGACTCGA AGTCGACGTG GGTGATCCTG CACCATAAAG TA - #TACGACCT     300    - AACTAAATTC CTCGAAGAGC ACCCCGGGGG CGAAGAAGTC CTGAGAGAAC AG - #GCCGGCGG     360    - TGACGCGACT GAAAACTTCG AAGACGTTGG CCATAGTACC GACGCTCGAG AA - #CTGTCGAA     420    - AACGTACATC ATCGGTGAGC TGCACCCGGA CGATCGTTCT AAAATCGCGA AA - #CCGTCCGA     480    #         511      TTCG ATATCAAGCT T    __________________________________________________________________________

I claim:
 1. A genetic precursor unit which comprises(a) a nucleotidesequence coding for a pre-form cytoplasmic cytochrome apo-protein (b) anucleotide sequence coding for an amino-terminal signal peptideoperationally linked to said nucleotide sequence coding for a pre-formcytoplasmic cytochrome apo-protein, said signal peptide capable ofdirecting said pre-form cytoplasmic cytochrome apo-protein to thecytoplasmic membrane of a cell for translocation into the periplasm ofsaid cell; and (c) a pho promoter operationally linked to the nucleotidesequences coding for said pre-form cytoplasmic cytochrome apo-proteinand said signal peptide.
 2. A genetic unit according to claim 1, whichis suitable for expression in the cytoplasmic region of an E.coli cell.3. A genetic unit according to claim 2, wherein said E.coli cellcomprises E.coli strain TB-1, F araΔ(lac-proAB)rpsφ80d lacZΔM15 hsdR17(rk+_(m) ⁺ k)! or N4830-1 f-suo his-ilv-galK-(λch1D-pg1)(λBam N+CI857H1)!.
 4. A genetic unit according to claim 1, wherein said cytoplasmiccytochrome comprises the soluble core domain of cytochrome b₅ of liverendoplasmic reticulum.
 5. A genetic unit according to claim 1, whereinsaid signal peptide comprises E.coli alkaline phosphatase.
 6. A methodof protein synthesis which comprises the genetic precursor unit setforth in claim 1, synthesizing said pre-form cytoplasmic cytochromeapo-protein in a cytoplasmic region of a cell, and translocating saidsynthesized pre-form cytoplasmic cytochrome apo-protein to theperiplasmic region of said cell, so as to permit constitution in saidperiplasmic region of a signal-processed cytoplasmic cytochromeapo-protein which can then be converted to a corresponding holo-protein.7. A method according to claim 6, wherein said cell comprises an E.colicell.
 8. A method according to claim 7, wherein said E.coli comprisesE.coli strain TB-1, F ara Δ(lac-proAB)rpsφ80d lacZΔM15 hsdR17 (rk+_(m) ⁺k)! or N4830-1 f-suo his-ilv-galK-(λch1D-pg1)(λBam N+CI857 H1)!.
 9. Amethod according to claim 6, which comprises initiation of transcriptionof said genetic precursor unit by cultivating a bacterial host in aphosphate-limited medium.
 10. A method according to claim 6, whichcomprises spectrophotometric or visual monitoring of a characteristiccolor change of host bacterial cells which is indicative of thesynthesis of said holo-protein or its fusion counterpart in saidbacterial cells.
 11. A method according to claim 6, which comprisestranslocation of a prosthetic group from the cytoplasmic region of thecell to the periplasmic region, for combination with thesignal-processed apo-protein in the periplasmic region to form theholo-protein.
 12. A method according to claim 11, wherein theapo-protein is translocated to the periplasmic region, followed bytranslocation of the prosthetic group, such that the apo-protein can actas a sink for the latter.
 13. A method according to claim 11, whereinthe apo-protein comprises the cytochrome b₅ soluble core, and theprosthetic material comprises the haem prosthetic group.
 14. A methodaccording to claim 6, wherein synthesis of the holo-protein is at alevel of at least 6 mg/liter of culture after five hours induction. 15.Apparatus for use in protein synthesis, which apparatus comprises:(a) asource of a bacterial cellular medium; and (b) a source of geneticmaterial comprising a genetic precursor unit of claim
 1. 16. Apparatusaccording to claim 15, wherein said bacterial medium comprises E.coli.17. An assay system for a signal peptidase, said assay systemcomprising:(a) the genetic precursor unit of claim 1; and (b) a signalpeptidase, a source thereof or a precursor therefor.
 18. An assay systemaccording to claim 17, wherein said genetic unit is suitable forexpression in the cytoplasmic region of an E.coli cell.
 19. An assaysystem according to claim 17, which further comprises a host medium forsaid genetic unit.
 20. An assay system according to claim 19, whereinsaid host medium comprises E.coli.
 21. An assay system according toclaim 20, wherein said E.coli comprises E.coli strain TB-1, FaraΔ(lac-proAB) rps φ80d lacZΔM15 hsdR17 (rk+_(m) ⁺ k)! or N4830-1 f-suohis-ilv-galK-(λch1D-pg1)(λBam N+CI857 H1)!.
 22. An assay systemaccording to claim 17, wherein said cytoplasmic cytochrome comprises thesoluble core domain of cytochrome b₅ of liver endoplasmic reticulum. 23.An assay system according to claim 17, wherein said signal peptidecomprises E.coli alkaline phosphatase.
 24. An assay system according toclaim 17, which further comprises means for monitoring the proteolyticcleavage of the amino terminal signal peptide from the apo-protein. 25.An assay system according to claim 24, wherein said monitoring meanscomprises means for separating the cleaved apo-protein, and means forspectrophotometrically monitoring the cleaved apo-protein followingsupplementation thereof with exogenous haem.
 26. An assay systemaccording to claim 25, wherein the pre-form apo-protein isphase-separated from the cleaved apo-protein.
 27. A method ofidentifying inhibitors to signal peptidase, which method comprisesproviding an assay system according to claim 17, introducing a testmaterial into cells of a host medium used in said assay system, andmonitoring the effect of said test material on a selected activity ofsaid signal peptidase.
 28. A method according to claim 27, whichinvolves spectrophotometrically monitoring the cleavage ofamino-terminal signal peptide from the apo-protein.
 29. A methodaccording to claim 27, wherein the test material comprises any ofconsensus signal peptide, at least one transition metal ion and acarboxy-modifying reagent.
 30. A vector as illustrated in any of FIGS. 1to
 3. 31. A DNA sequence as illustrated in any of FIGS. 4 to 6 (SEQ IDNOS 1 to 3 respectively).